Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments.

Enzymatic cleavage of carotenoids

1. 1.|Carotene 15,15′-dioxygenase (EC 1.13.11.21) has been isolated from the intestine of guinea pig and rabbit and purified 38- and 30-fold, respectively, but subjecting the intestinal homogenate to protamine sulfate treatment, (NH4)2SO4 fractionation and acetone precipitation. 2. 2.|The guinea pig enzyme showed a pH optimum at 8.5, an optimum substrate concentration of 200 nmoles of β,β-carotene per 25 ml of reaction mixture, an apparent Km with β,β-carotene as substrate of 9.5 · 10−6 M and a V 3.3 nmoles of retinal formation/mg protein per h. The reaction was linear upto 3 h and the reaction rate increased linearly with increase in enzyme protein concentration. The enzyme was activated by GSH and Fe2+ and inhibited by sodium dodecylsulfate, sulfhydryl binding and iron chelating agents. 3. 3.|The reaction catalysed by guinea pig enzyme was strictly stoichiometric. 4. 4.|Rabbit enzyme showed a close similarity with guinea pig enzyme with respect to time course, optimum substrate concentration, activation by Fe2+ and GSH, inhibition by sodium dodecylsulfate, iron chelating and sulfhydryl binding agents. However, it showed a slightly lower pH optimum (pH 7.8). 5. 5.|The enzyme from guinea pig and rabbit showed remarkable similarity with respect to cleavage of carotenoids. The enzyme from both the species was more specific for β,β-carotene but could also cleave a number of other carotenoids at the 15,15′-double bond. 6. 6.|10′-Apo-β-carotenal and 10′-apo-β-carotenol were readily cleaved compared with other apo-β-carotenals and apo-β-carotenols tested. 7. 7.|It has been conclusively shown for the first time that mono-ring substituted carotenoids are also cleaved at the 15,15′-double bond.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

Varied Immune Response to FVIII: Presence of Proteolytic Antibodies Directed to Factor VIII in Different Human Pathologies

The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation of the complement and its activation, and activation of effector cells. In addition to this plethora of functions, antibodies are capable of expressing enzymatic activity. Antibodies with catalytic function are a result of the productive interplay between the highly evolved machinery of the immune system and the chemical framework used to induce them (antigens). Catalytic antibodies are immunoglobulins with an ability to catalyze the reactions involving the antigen for which they are specific. Catalytic immunoglobulins of the IgM and IgG isotypes have been detected in the serum of healthy donors. In addition, catalytic immunoglobulins of the IgA isotype have been detected in the milk of healthy mothers. Conversely, antigen-specific hydrolytic antibodies have been reported in a number of inflammatory, autoimmune, and neoplastic disorders. The pathophysiological occurrence and relevance of catalytic antibodies remains a debated issue. Through the description of the hydrolysis of coagulation factor VIII as model target antigen, we propose that catalytic antibodies directed to the coagulation factor VIII may play a beneficial or a deleterious role depending on the immuno-inflammatory condition under which they occur.

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.

Photocytotoxic 3d-Metal Scorpionates with a 1,8-Naphthalimide Chromophore Showing Photoinduced DNA and Protein Cleavage Activity

Ternary 3d-metal complexes of formulation [M(Tp(Ph))(py-nap)](ClO4)(1-3), where M is Co(II) (1), Cu(II) (2), and Zn(II) (3); Tp(Ph) is anionic tris (3-phenylpyrazolyl)borate; and py-nap is a pyridyl ligand with a conjugated 1,8-naphthalimide moiety, have been prepared from the reaction of metal perchlorate with KTp(Ph) and py-nap in CH2Cl2. The complexes have been characterized from analytical and physicochemical data. The complexes are stable in solution as evidenced from the electrospray ionization mass spectrometry data. The complexes show good binding propensity with calf thymus (CT) DNA, giving binding constant (K-b) values of similar to 10(5) M-1 and a molecular ``light-switch'' effect that results in an enhancement of the emission intensity of the naphthalimide chromophore on binding to CT DNA. The complexes do not show any hydrolytic cleavage of DNA. They show poor chemical nuclease activity in the presence of 3-mercaptopropionic acid or hydrogen peroxide (H2O2). The Co(II) and Cu(II) complexes exhibit oxidative pUC19 DNA cleavage activity in UV-A light of 365 rim. The Zn(II) complex shows moderate DNA photocleavage activity at 365 nm. The Cu(II)complex 2 displays photoinduced DNA cleavage activity in red light of 647.1 nm and 676 rim and near-IR light of >750 rim. A mechanistic studyin UV-A and visible light suggests the involvement of the hydroxyl radical as the reactive species in the DNA photocleavage reactions. The complexes also show good bovine serum albumin (BSA) protein binding propensity, giving K-BSA values of similar to 10(5) M-1. Complexes 1 and 2 display significant photoinduced BSA cleavage activity in UV-A light. The Co(II) complex 1 shows a significant photocytotoxic effect in HeLa cervical cancer cells on exposure to UV-A light of 365 nm, giving an IC50 value of 32 mu M. The IC50 value for the py-nap ligand alone is 41.42 mu m in UV-A light. The IC50 value is >200 mu M in the dark, indicating poor dark toxicity of 1. The Cu(II) complex 2 exhibits moderate photocytotoxicity and significant dark toxicity, giving IC50 values of 18.6 mu m and 29.7 mu m in UV-A light and in the dark, respectively.

New ternary copper(II) complexes of L-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of alpha-amino acid having polypyridyl bases of general formulation [Cu(L-ala)(B)(H2O)](X)(1-4), where L-ala is L-alanine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2',3'-f] quinoxaline (dpq, 4), and X = ClO4-/NO3- are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN3O2 coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near - 100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 >>>1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Substrate Selectivity and Molecular Adaptation in the Outer Membrane Proteases of Yersinia pestis and Salmonella enterica

Proteolysis is important in bacterial pathogenesis and colonization of animal and plant hosts. In this work I have investigated the functions of the bacterial outer membrane proteases, omptins, of Yersinia pestis and Salmonella enterica. Y. pestis is a zoonotic pathogen that causes plague and has evolved from gastroenteritis-causing Yersinia pseudotuberculosis about 13 000 years ago. S. enterica causes gastroenteritis and typhoid fever in humans. Omptins are transmembrane β-barrels with ten antiparallel β-strands and five surface-exposed loops. The loops are important in substrate recognition, and variation in the loop sequences leads to different substrate selectivities between omptins, which makes omptins an ideal platform to investigate functional adaptation and to alter their polypeptide substrate preferences. The omptins Pla of Y. pestis and PgtE of S. enterica are 75% identical in their amino acid sequences. Pla is a multifunctional protein with proteolytic and non-proteolytic functions, and it increases bacterial penetration and proliferation in the host. Functions of PgtE increase migration of S. enterica in vivo and bacterial survival in mouse macrophages, thus enhancing bacterial spread within the host. Mammalian plasminogen/fibrinolytic system maintains the balance between coagulation and fibrinolysis and participates in several cellular processes, e.g., cell migration and degradation of extracellular matrix proteins. This system consists of activation cascades, which are strictly controlled by several regulators, such as plasminogen activator inhibitor 1 (PAI-1), α2-antiplasmin (α2AP), and thrombin-activatable fibrinolysis inhibitor (TAFI). This work reveals novel interactions of the omptins of Y. pestis and S. enterica with the regulators of the plasminogen/fibrinolytic system: Pla and PgtE inactivate PAI-1 by cleavage at the reactive site peptide bond, and degrade TAFI, preventing its activation to TAFIa. Structure-function relationship studies with Pla showed that threonine 259 of Pla is crucial in plasminogen activation, as it prevents degradation of the plasmin catalytic domain by the omptin and thus maintains plasmin stability. In this work I constructed chimeric proteins between Pla and Epo of Erwinia pyrifoliae that share 78% sequence identity to find out which amino acids and regions in Pla are important for its functions. Epo is neither a plasminogen activator nor an invasin, but it degrades α2AP and PAI-1. Cumulative substitutions towards Pla sequence turned Epo into a Pla-like protein. In addition to threonine 259, loops 3 and 5 are critical in plasminogen activation by Pla. Turning Epo into an invasin required substitution of 31 residues located at the extracellular side of the Epo protein above the lipid bilayer, and also of the β1-strand in the N-terminal transmembrane region of the protein. These studies give an example of how omptins adapt to novel functions that advantage their host bacteria in different ecological niches.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Photocytotoxic Oxovanadium(IV) Complexes Showing Light-Induced DNA and Protein Cleavage Activity

Oxovanadium(IV) complexes [VO(L)(B)]Cl-2 (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline(phen),dipyrido[3,2-d:2',3'-f]quinoxaline(dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells, The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON5 coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III)couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M-1. The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor ``chemical nuclease'' activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells giving IC50 value of 17 mu M in visible light(IC50 = 175 mu M in dark).

The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn(2+) and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg(2+) alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg(2+), PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp(122), Asp(222), and Glu(220)) together with immunoprecipitation assays provided compelling evidence to link both the Mg(2+)- and Mn(2+) and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations.